The present invention relates to methods and compostions for the detection of Legionnaires' desease and its causative agent, Legionella pneumophila, in an infected host. More specifically the methods described herein rely on the generation of immunologic antigen products specific for the detection of Legionella pneumophila-directed antibodies. These Legionella pneumophila antigen products are derived from a recombinant DNA clone bank comprised of L. pneumophila genes and expressed in an E. coli cloning host.
Current methods for the diagnosis of Legionnaires' disease in infected hosts are somewhat unreliable and generally result in a high level of falsely positive or falsely negative detections. Part of the problem in identifying the disease in a suspected carrier has been an inability to isolate the causative agent from the host. Successful isolation of the causative agent is required for conventional diagnosis of most bacterial infections. Alternative approaches to Legionnaires' disease detection include: (1) the staining characteristics and microscopic morphology of the L. pneumophila microorganism; (2) direct fluorescent staining of the microorganism with specific antiserum; and (3) the use of crude L. pneumophila antigens for the detection of circulating antibodies in an infected host.
Detection of Legionnaires' disease through the use of non-specific staining procedures has not proven to be a particularly sound basis for diagnosis. Although, L. pneumophila is a gram-negative organism, it stains poorly by conventional gram-staining techniques. Differential staining, where samples stained by the conventional gram method are compared to Lugol staining has been used to identify L. pneumophila organisms with varied success. Other non-specific stains have been tried, including Dieterle silver, Giemsa, methylene blue, and Gimenez, also with varied success (for a general discussion see Lattimer, G., Legionnaires' Disease, Marcel Decker, Inc., New York, 1981, pp. 65-81). These stains may be suggestive of the presence of L. pneumophila but are by no means definitive. They are useful only when no other bacteria are present or when examining infected tissue directly.
One approach to the identification of the organism is provided by direct fluorescent antibody staining with antisera directed to L. pneumophila antigens. Generally speaking, this method involves the generation of Legionella-directed antibodies in hyperimmune rabbit antisera followed by FITC conjugation of the antibodies. The FITC-conjugated anti-Legionella rabbit antisera is then reacted with slide-fixed suspected L. pneumophila-containing tissue, aspirate, or sputum samples. Some promising results have been demonstrated by this technique but caution must be exerted. First, most antisera are raised in rabbits with the aid of Freund's adjuvant, which contains mycobacterium. Therefore, the resulting rabbit Ig will often react with mycobacterium present in patient's sputum or pleural fluids. Second, antibodies may cross-react with a significant number of clinical isolates of Bacteroides fragilis.
Therefore, while the direct immuno-fluoresence test remains the method of choice for quick identification of the L. pneumophila organism it still suffers from many of the above-mentioned drawbacks. The technique requires an active sputum, pleural, or tissue sample containing the bacterium, which may not be available at the earlier stages of the disease, when rapid detection is of primary importance. In addition, the sensitivity of the direct immunofluorescence test is low, and therefore only about 60-75% of active cases are detected.
Another approach to the diagnosis of Legionnaires' disease, which is now the accepted standard, involves detecting circulating antibodies formed in the infected host in response to antigens present on the surface of the L. pneumophila bacterium. This has been accomplished by first inactivating the L. pneumophila organisms by either formaldehyde or steam killing and using the fixed bacteria in an indirect immunofluorescent assay for the detection of reacting antibodies in the suspected host. The assay requires both acute and convalescent serum samples and is not interpretable when performed on single serum samples during infection because of high background levels of non-specific antibodies. It has been suggested that the primary reason for the high level of nonspecific reactions in this diagnostic procedure is the presence of L. pneumophila cellular antigens that cross-react with other common bacteria to which humans are exposed. To produce a specific immunoassay, it would therefore be essential to separate specific from cross-reactive L. pneumophila antigens.
Therefore, in light of the above drawbacks in present Legionnaires' disease diagnosis, a technique which is both rapid, sensitive, and specific is needed in order to detect the presence of the disease early on, and diagnose it correctly and reproducibly. One such approach would be to utilize recombinant DNA technology whereby genes specific to L. pneumophila are used to specifically detect, by nucleic acid hybridization, the presence of L. pneumophila organisms in clinical samples. Owing to the specificity inherent in nucleic acid hybridization, such a technique should be capable of detecting the bacterium at extremely low levels in, for instance, sputum or pleural samples. A second possible detection method, which is provided by the present invention, is availed by the successful isolation of individual antigen molecules from L. pneumophila. Such antigens could then be used either directly to detect circulating antibodies, or to prepare highly specific antibodies or hybridomas. Antigens or antibodies prepared in this manner could be selected so as to be highly specific.